There are known in vivo enzymes having serine, threonine or tyrosine residue at a specific site such as an active center. The enzymatic activity of these enzymes is controlled by phosphorylating, i.e. monoesterification by a phosphate, or dephosphorylating hydroxyl group in these residues by an enzyme called kinase. Also, there are known enzymes whose enzymatic activity is controlled by phosphorylating or dephosphorylating a nitrogen in lysin, arginine or histidine, or a carboxyl group in aspartic acids or glutamic acids.
One of the examples of the metabolic systems which are controlled by the aforementioned phosphorylation-dephosphorylation is a system of suppressing synthesis of glycogen and decomposing the same. This metabolic system is primarily cascade-controlled by the phosphorylation-dephosphorylation.
A recent study has elucidated that the phosphorylation-dephosphorylation plays a significant role in disease-related metabolic systems.
For instance, it is said that one of the causes of cell carcinogenesis is abnormality in the phosphorylation-dephosphorylation. Specifically, progress and stop of cell cycle are controlled by phosphorylation or dephosphorylation of various enzymes, i.e. proteins. Cycline and cycline-dependent kinase (CDK) are relevant factors in the phosphorylation. In case where the mechanism relating to cycline and CDK is impaired, phosphorylation or dephosphorylation may be uncontrollable, thereby abnormal proliferation of cells is triggered.
In addition to the above, facts are known that protein kinase C is related with degranulation of histamine causative of allergic disorders such as atopic dermatitis and pollen allergy, and that phosphorylated tau-protein is causative of neurofibrillary tangle in the brains of Alzheimer's patients.
In view of the above, comprehending which enzymes, i.e. proteins, in biological samples are phosphorylated or dephosphorylated could provide useful measures not only in investigating expression of genes in living tissue cells and evaluating the enzymatic activity of the cells, but also in diagnosing diseases or medical treatment.
The conventional methods for identifying phosphorylated proteins or dephosphorylated proteins have various drawbacks.
For instance, while an enzyme immunoassay is advantageous in analyzing a target protein sample of a very small amount, it is difficult to obtain antibodies of the target protein of a sufficient amount. Further, in case that the level of the target protein is several kDa or lower, it is impossible to prepare an antibody that is securely bonded to a site in the protein where phosphorylation occurs.
There is proposed a method for detecting a protein specifically bonded by a phosphoric acid with use of a phosphoric acid labeled with a radioactive isotope 32P. However, special attention should be paid in handling radioactive isotopes, and appropriate administration and disposal of waste liquid of the radioactive isotopes are required.
There is proposed an idea of applying two-dimensional electrophoresis in view of the fact that electric charges are differentiated between phosphorylated proteins and dephosphorylated proteins. However, it is extremely difficult to identify the spot of a phosphorylated or dephosphorylated protein in analyzing a sample derived from a living organism, because the sample contains a variety of proteins. Furthermore, use of a radioactive isotope to identify the spot involves the aforementioned problems.
The document, Morio YASHIRO, et al, ┌Preparation and Study of Dinuclear Zinc (II) Complex for the Efficient Hydrolysis of the Phosphodiester Linkage in a Diribonucleotide┘, Journal of the Chemical Society, Chemical communications, pp. 1793-1794 (1995), recites a zinc complex. The zinc complex has a function that two zinc ions act on the phosphoric acid group in dinucleotide and dissociate the dinucleotide. However, the function of the zinc complex disclosed in the document is merely a catalyst. The document does not disclose the ability of the zinc complex to bond coordinately to a phosphoric acid group. The experiments conducted by the inventors of the present invention reveal that a dissociation constant of the zinc complex to a phosphoric acid group sandwiched by two nucleotides, namely a phosphoric diester group, is extremely high. In other words, the zinc complex has a low coordinatability to a phosphoric diester moiety.
Further, the document, Hidekazu ARII, et al., ┌A novel diiron complex as a functional model for hemerythrin┘, Journal of Inorganic Biochemistry, 82, pp. 153-162 (2000), recites an iron complex having a structure analogous to the structure of the zinc complex. The iron complex, however, is a product synthesized as a model of hemerythrin, namely a carrier protein carrying oxygen molecules. As is the case with the above mentioned document, this document neither discloses nor suggests coordinate bond of the iron complex to a phosphoric monoester moiety at all.
The inventors of the present invention have already developed a method for identifying peptide and the like having a phosphoric acid monoester group. In the method, a complex which specifically bonds coordinately to an anionic substituent such as a phosphoric acid monoester group is used, and mass spectra of samples with and without the aforementioned complex are compared, thereby information on the compound which the phosphoric acid monoester group bonded can be obtained. That is, since values of molecular ion peaks between the compound with the complex and the compound without the complex are different depending on the existence of the complex, the molecular weight of the compound having the phosphoric acid monoester group can be identified.
However, except for pure samples, i.e. purified samples, when a sample including a plurality of compounds is analyzed, a molecular ion peak to be detected may not be identified even if a chart is enlarged. Indication of ion peaks may vary depending on distribution of isotopes of atoms configured of a compound. Therefore, indication of molecular ion peaks is different between the compound with the complex and the compound without the complex, and it may be difficult to identify the peaks.